Alcohol dependence is a chronic, relapsing disorder characterized by dysregulated mood states and compulsive alcohol intake. Evidence suggests that dysregulated mood states (e.g., depression, anxiety) increase the likelihood of relapse to alcohol drinking, even following long periods of abstinence. Therefore, understanding the neural mechanisms that underlie dysregulated mood states will provide insight into new pharmacological targets for the treatment of alcohol dependence and comorbid disorders. To this end, we are using Gi-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) to assess the contribution of the periaqueductal gray area (PAG) to dysregulated mood states and compulsive alcohol intake. Results thus far suggest that the dorsal portion of the PAG modulates excessive alcohol self-administration, whereas the ventral portion modulates alcohol self-administration and enhanced anxiety states during alcohol withdrawal. Future work will assess the contribution of specific PAG neuronal populations to these behaviors. We tested the effects of oxytocin on compulsive alcohol drinking using systemic injections and intranasal delivery. Rats were trained to lever press for access to alcohol (i.e., operant conditioning). Some of the animals are made dependent on alcohol via chronic, intermittent alcohol vapor exposure. This results in a dramatic increase in alcohol self-administration. Using this model, we observed that systemic injections and intranasal delivery of oxytocin substantially reduced alcohol consumption in dependent but not in nondependent rats. Histological analysis determined that dependent rats (during acute alcohol withdrawal) have increased cellular activity, particularly in the central amygdala, compared to nondependent rats. We are currently working in collaboration with an electrophysiology team to determine the cellular changes induced by oxytocin in the central amygdala, as well as providing data as the basis for a clinical trial of oxytocin in alcohol dependent humans, to be conducted in collaboration with the NIAAA. Ghrelin is an orexigenic, gastric hormone that has been implicated in substance use and dependence. To further explore the role of the ghrelin system in relation to drug and alcohol abuse, we developed at NIDA, in collaboration with the NIDA optogenetic and transgenic core, a transgenic ghrelin receptor knockout rat using CRISPR. RNAscope imaging confirmed deletion of the ghrelin receptor. We observed that the ghrelin receptor knockout rats weigh significantly less than wild-type rats, consume less chow, and do not increase chow consumption in response to a ghrelin challenge. Future studies will investigate the role of the ghrelin system in drug and alcohol dependence, and related behaviors. The translational nature of this work is significant, as we are working in collaboration with a clinical laboratory at the NIAAA to provide rodent data to inform clinical studies on the role of the ghrelin system in drug and alcohol dependence. Additional ongoing studies are investigating the role of sleep/wake state in alcohol dependence. Hypocretin is a neuropeptide that has been associated with both stress and arousal states, as well as drug and alcohol seeking behavior. Interestingly, clinical studies indicate associations between sleep dysfunction and drug abuse and/or relapse. Electroencephalogram data reveal that non-REM and REM sleep onset latency are both predictors of relapse. Pilot studies in our laboratory utilizing radio telemetry to measure sleep/wake state show altered sleep architecture during acute alcohol withdrawal. Given the preclinical efficacy of HCRT-receptor antagonist treatment of either alcohol dependence or sleep disorders, a viable pharmacological target uniting treatment of both sleep dysfunction and substance abuse disorders clinically in the same individual may be found within the HCRT stress/arousal system. Opioid misuse is at historically high levels in the United States. We developed a model of opioid dependence by inhalation that does not require surgery. Rats were trained to perform an operant response (nosepoke) to receive 10 s of vaporized sufentanil, a potent opioid, in 2 h daily sessions. Rats readily and concentration-dependently self-administered vaporized sufentanil. The reinforcing properties of sufentanil was found to involve -opioid receptors. Serum sufentanil concentrations significantly correlated with the number of sufentanil vapor deliveries. Rats that were given long access (LgA; 12 h/day) but not short access (ShA; 1 h/day) to vaporized sufentanil escalated their drug intake over time and exhibited somatic signs of opioid withdrawal and mechanical hypersensitivity. After 6 months of forced drug abstinence, LgA rats returned to pre-escalation baseline levels of responding for sufentanil and mechanical sensitivity. Upon subsequent re-escalation (i.e., after the return to extended access to sufentanil vapor), LgA rats again developed somatic signs of withdrawal and mechanical hypersensitivity. These findings demonstrate that the operant sufentanil vapor self-administration model has both face and construct validity, and therefore will be useful for investigating the neurobiological basis of opioid addiction. We tested the hypothesis that neural circuits mediating reward and negative emotional states have a key role in learning that contributes to the maintenance of compulsive opioid seeking. We found that when given alone, cues previously paired with withdrawal-induced aversive states can increase operant responding for heroin, promote reinstatement of lever pressing behavior following extinction, and alter pain sensitivity in heroin dependent rats. Using functional magnetic resonance imaging in collaboration with the Neuroimaging Research Branch, we identified several cortical and subcortical brain regions that are activated by presentation of these cues in the absence of heroin, including extended amygdala and hypothalamic stress circuitries. These results point to novel targets for therapeutic intervention, specifically brain circuits of negative emotional learning. Follow up studies are using molecular imaging (e.g., immunohistochemistry and in situ hybridization) in collaboration with the Morales lab and Histology Core to characterize these circuits, which represent a potentially powerful source of motivation in addiction. Future studies will use (DREADDs technology to examine the functional role of the extended amygdala and hypothalamic stress circuitries in opioid dependence. Cocaine abuse affects approximately 1.7 million individuals nationwide per year and is characterized by patterns of excessive drug seeking and taking, including a preoccupation with obtaining the drug, repetitive seeking and taking of the drug, and a loss of control over drug intake. Compulsive-like cocaine taking and reinstatement of drug seeking (a model of drug relapse) occur in part through neuroadaptations of brain stress systems that mediate negative emotional states implicated in motivational processes required for maintaining the dependent drug state. The goal of the current research is to characterize the role of neuropeptides, including hypocretin and dynorphin in the mediation of cocaine intake and the reinstatement of drug seeking following extinction. To date, we have shown hypocretin- or dynorphin-receptor antagonism within target extended amygdala brain regions results in attenuation of drug taking and seeking. Futhermore, we have shown long-term silencing of the hcrt gene via adeno-associated viral vectors attenuates cocaine self-administration in dependent animals. Future studies are investigating the use of FDA-approved hypocretin-receptor antagonists in further elucidating the conditional role of hypocretin in cocaine-seeking.